1. Protein NMR Structure Determination Institute of Biomedical Sciences
Yuan-Chao Lou
羅元超
Institute of Biomedical Sciences
Academia Sinica

2. Outline Protein NMR Structure Determination
(1). Nuclear Overhauser Effect (NOE)
(2). Dihedral Angle and J Coupling Constant
(3). H-bond and Amide Proton Exchange Rate
B. Software for Structure Determination
(1). X-PLOR
(2). CYANA
C. New Techniques in Protein NMR
(1). Residual Dipolar Coupling (RDC)
(2). Transverse Relaxation-Optimized Spectroscopy (TROSY)

3. Nuclear Overhauser Effect
Nuclear Overhauser effect (NOE) was discovered by Albert Overhauser in He found that saturation of the electron magnetic resonance in a paramagnetic system would cause the nuclear resonance intensity to be enhanced. A similar effect occurs between nuclei. It is much smaller, but still observable.
NOE enhancements between nuclei are due to nuclei’s dipole-dipole interactions (through-space) and are correlated with the inverse sixth power of the internuclear distance.
 : permeability constant
h : Planck’s constant
 : magnetogyric ratio
c : rotational correlation time
 : larmor frequency
J() = 2c / (1 + 2c2)

4. Dependence of NOE on Molecular Motion
NOE = 0 when c = (If I = S)
A peptide which contains around 10 residues can only get very weak NOE signals.

5. Dependence of NOE on Distance
NOE / NOEstd
= rstd6 / r 6
The shorter the distance, the stronger the NOE effect. Basically, we can observe the NOE cross peak between two protons if their distance is smaller than 5 Å. And we can get the distance information from the intensity of the NOE cross-peaks.

6. Protein NMR Spectra

7. Why Bother with 15N Incorporation ?
15N-HSQC

8. Why Bother with 13C Incorporation ?
13C-HSQC

9. 2D NOESY spectrum

10. Dihedral Angles Cγ χ2 Cβ χ1 Cα N ψ Ψ ω N C’ O
Karplus Equations:

11. Dihedral Angles and J Coupling Constants of Different Secondary Structures
Dihedral Angles (deg)
3JHNα(HZ)
≧ 9
≦ 4 β βp
α-helix
310-helix
π-helix
Polyproline I
Polyproline II
Polyglycine II ψ
-139
-119
-57
-49
-83
-78
-80 Ψ
+135
+113
-47
-26
-70
+158
+149
+150 ω
-178
180
Adapted from G. N. Ramachandran and V. Sasisekharan, Adv. Protein Chem. 23, (1968); IUPAC-IUB Commission on biochemical Nomemclature, Biochemistry 9, (1970).

12. Extracting J Coupling Constants from 1D spectra
3JHNα

13. Extracting J Coupling Constants from 2D DQF-COSY
3JHNα

14. Extracting J Coupling Constants from 3D HNHA Spectra
Scross/Sdiag = -tan2(2JHNa )

15. Getting Dihedral Angle Restraints from Searching a Database by TALOS
TALOS is a database system for empirical prediction of phi (f) and psi (y) backbone dihedral angles using a combination of five kinds (Ha, Ca, Cb, C’, N) of chemical shift assignments for a given protein sequence.

16. Getting H-bond Restraints from Amide Proton Exchange Rate
Exchangeable Protons:
－NH ; －OH ; －SH
Amide protons that are protected by H-bonds or hydrophobic residues exhibit lower exchange rate

17. Proton Distances, Coupling Constants, and Amide Proton Exchange rate in a-helix
Parameter
dαN(i,i+1)
dαN(i,i+2)
dαN(i,i+3)
dαN(i,i+4)
dNN(i,i+1)
dNN(i,i+2)
dβN(i,i+1)
dαβ (i,i+3)
3JHNα(HZ)
HN exchange rate
α-helix
3.5
4.4
3.4
4.2
2.8
(≦4)
slow
310-helix
3.4
3.8
3.3
(>4.5)
2.6
4.1
(≦4)
slow C’ N’
The first four residues in the α-helix and the first three residues in the 310-helix will have fast amide proton exchange rates.

18. Proton Distances, Coupling Constants, and Amide Proton Exchange rate in b-strand
Parameter
dαN(i,i)
dαN(i,i+1)
dNN(i,i+1)
dβN(i,i+1)
dαα (i,j)
dαN (i,j)
dNN (i,j)
3JHNα(HZ)
NH exchange rate β
2.8
2.2
4.3
2.3
3.2
3.3
(≧9)
slow βP
2.8
2.2
4.2
4.8
3.0
4.0
(≧9)
slow N’ C’ N’ C’
dαα(i,j), dαN (i,j) and dNN (i,j) refer to interstrand distances.
Every second residue in the flanking strand will have slow amide proton exchange rates

19. Observed NOEs in Secondary Structures
dNN(i,i+1)
dαN(i,i+1)
dαN(i,i+3)
dαβ (i,i+3)
dαN(i,i+2)
dNN(i,i+2)
dαN(i,i+4)
3JHNα(HZ)
α-helix
310-helix
β-strand
The thickness of the lines is an indication of the intensity of the NOEs
The values of J coupling are approximate.

20. NOESY and TOCSY : Amide to a RegionH O

21. The Definition of b -sheet of TC1
The b-sheet structure of Tc1 can be defined based on NOEs and amide proton exchange rate

22. Summary of the amide proton exchange rates, 3JNHα coupling constants, NOE connectivities, chemical shift index, and the derived secondary structures

23. Protein NMR Structure Determination
Protein in solution
~0.5 ml, 2 mM concentration
Sample preparation:
cloning,
protein expression
purification,
characterization,
isotopic labeling.
Distances between
protons (NOE),
Dihedral angles(J
coupling), H-bond
(Amide-proton
exchange rate ),
RDC restraints
NMR spectroscopy
1D, 2D, 3D, …
Secondary
structure of
protein
Sequence-specific
Resonance assignment
Extraction of Structural
information
Structure calculation
Final 3D
structures
Structure refinement

24. The Completeness of Assignment is an Determinant for NOESY Assignment
residue N C C C
other Q1
(8.379)
(4.111)
(2.834, 2.302)
C, (2.644, 2.644) D2
(7.959)
(4.746)
(3.154, 3.154) W3
(9.602)
(5.635)
(3.526, 3.317)
C1, (7.384); C3, (8.290); C2, (7.286); C2, (7.308); C3, (6.811); N1, (10.193) E4
(8.707)
(3.782)
(2.021, 2.021)
C, (2.422, 2.200) T5
(8.910)
(3.942)
(3.739)
C2, (1.248) F6
(8.796)
(4.228)
(3.615, 3.171)
C1, (7.165); C2, (7.165); C1, (7.024); C2, (7.024); C, (6.834) Q7
(8.118)
(3.647)
(1.193, 1.193)
C, (1.851, 1.851); N2, (6.195, 4.472) K8
(7.449)
(4.036)
(1.807, 1.770)
C, (1.487, 1.487); C, (1.710, 1.710); C, (2.944, 2.944) K9
(8.261)
(4.167)
(1.626, 1.626)
C, (1.090, 1.090); C, (1.398, 1.398); C, (2.882, 2.882)
H10
(7.803)
(4.846)
(2.767, 2.000)
C2, (6.786); C1, (8.755)
L11
(8.311)
(5.406)
(2.156, 2.156)
C, (1.788); C1, (1.103); C2, (1.103)
T12
(8.237)
(5.003)
(3.775)
C2, (1.220)
D13
(8.271)
(4.874)
(3.060, 2.766)
T14
(8.106)
(4.802)
(4.067)
C2, (0.947)
K15
(8.239)
(3.623)
(1.440, 1.440)
C, (0.736, 0.405); C, (1.423, 1.423); C, (2.741, 2.741)
K16
(7.904)
(4.277)
(1.680, 1.680)
C, (1.234, 1.234); C, (1.545, 1.545); C, (2.953, 2.953)
V17
(6.052)
(3.325)
(1.441)
C1, (0.405); C2, (0.105)
K18
(8.665)
(4.488)
(1.886, 1.886)
C, (1.548, 1.548); C, (1.748, 1.748); C, (3.062, 3.062)
C19
(8.066)
(3.692)
(3.027, 2.339)
D20
(8.880)
(4.453)
(3.056, 2.905)

25. NOESY Spectra of hPAP
600 MHz
800 MHz

26. NOESY Assignment
10A
10B
10C
Intra-residual NOE ( |i-j| = 0)
Sequential NOE ( |i-j| = 1)
Medium-range NOE ( |i-j| < 5)
Long-Range NOE ( |i-j| ≥ 5) C 3B 1 D A B 1B 1C 1D 2 B 3 B 4 2B 5 D C 6 C 11 B B 1 10 7 A A 2 9 8 B 8 3 B 9 4 7 B C 10 A 5 6 11 1A

27. Three-Dimensional Structure Determination by Simulated Annealing using X-PLOR or CNS Program
dih
noe
vdw
improper
angle
bond
total E + =
Keep the correctness of protein geometry
The energy terms of experimental data

28. Automated NOESY Assignment and Structure Calculation
Protein Sequence
Chemical shift list
Positions and volumes of NOESY cross peaks
Automated methods are
- much faster
- more objective
Find new NOE assignments
Problems may arise because of
- imperfect input data
- limitation of the algorithms used
Structure Calculation
Iterative process : All but the first cycle use the structure from the preceding cycle.
Evaluate Assignments
Finish NOESY Assignment
3D NMR Structure
The first cycle is important for the reliability of the method.

29. Algorithms Used by CYANA
Network-Anchoring can find the new NOE assignment correctly.

30. Structural Statistics of the Best 20 Structures

31. Ramachandran Plot

32. 3D Structure Determination of RNase 3 from Rana catesbeiana

33. New Techniques in Protein NMR
(1). Residual Dipolar Coupling (RDC)
(2). Transverse Relaxation-Optimized Spectroscopy (TROSY) or
NOE, dihedral angle and H-bond are short-range restraints and have limitations for some structure determination, like extended structures or multiple-domain structure. RDC is a novel restraint and provides global structure information.
TROSY, which was developed by K. Wüthrich, can select one fourth of the signals that relax more slowly than the others. The Utilization of TROSY techniques push the size limit of NMR spectroscopy to 30~50 kDa.

34. Residual Dipolar Coupling (RDC)
Residual dipolar couplings arise from dipole-dipole interactions between nuclei. In aqueous solution, the isotropic orientation of the molecules average out the dipolar couplings. However, in oriented media, the molecular tumbled anisotropically. The order of 10-4 to 10-3 of anisotropy tuned the dipolar coupling constant to be a residual value of few Hz, which are well detectable by NMR spectroscopy.
Values of static dipolar coupling constant of two-spin systems in protein backbone.

35. Residual Dipolar Coupling (RDC)
The residual dipolar coupling between two spins A and B are given by :
<DAB> = - C(Bo) [ a(3cos2 -1) + 3/2 r(sin2 cos2) ]
where
C(Bo) = S(Bo2/15kT)[AB h/(4p2rAB3).
A and B are gyromagnetic ratios of A and B.
rAB is the distance between A and B.
So, Bo , DAB
S (order parameter) , DAB

36. Alignment Media Phages (Pf1, fd, TMV) (Zweckstetter, JBNMR)
Bicelles (Sanders & Schwonek, Biochemistry, 1992; Ottiger&Bax, JBNMR 1998)
Polyacrylamide gels (Tycko,JBNMR; Grzesiek JBNMR; Chou, JBNMR)
Paramagnetic tagging (Opella , Griesinger, Byrd)
CPBr/hexanol (Barrientos, J. Mag. Res, ~2000)
C12E5/hexanol (Ruckert&Otting, JACS 2000)
Cellulose crystallites (Matthews, JACS, ~2000)

37. Alignment of Molecules in Anisotropic Solutions
The most-used media for RDC measurement are :
(a). Phospholipid bicelles and (b). Filamentous phage

38. 15N-IPAP HSQC for HN RDC values
1H Chemical Shift (ppm)
Isotropic solution
+ 5.3 mg/ml Pf1
15N Chemical Shift (ppm)
JNH
JNH + DNH

39. 3D HNCO for C’N RDC values
1H Chemical Shift (ppm)
Isotropic solution
+ 5.3 mg/ml Pf1
13C Chemical Shift (ppm)
JC’N + DC’N
JC’N

40. Structure Refinement with RDC Restraints
<DAB>(q,f) = Da [ a(3cos2 -1) + 3/2 Dr(sin2 cos2) ] X Y Z

41. Transverse Relaxation-Optimized Spectroscopy (TROSY)
(a). None-decoupled HSQC
(b). Decoupled HSQC
(c). TROSY-HSQC
15N 1H
Main relaxation source for 1H and 15N: dipole-dipole (DD) coupling and, at high magnetic fields, chemical shift anisotropy (CSA).
Different relaxation rates (line width) for each of the four components of 15N-1H correlation.
The narrowest peak (the blue peak) is due to the constructive canceling of transverse relaxation caused by chemical shift anisotropy (CSA) and by dipole-dipole coupling at high magnetic field.
TROSY selectively detect only the narrowest component (1 out of 4).

42. DD + CSA DD – CSA DD + CSA DD – CSA
Interference between DD and CSA Relaxation
DD + CSA
(large) (large)
DD – CSA
At High Magnetic Field
(TROSY line-narrowing effect)
DD + CSA
(large) (small)
(B) At Low Magnetic Field
(almost no TROSY
line-narrowing effect)
DD – CSA
(large) (small)
DD relaxation is field-independent. However, CSA relaxation  B02,
therefore at high magnetic fields, CSA relaxation can be comparable to
DD relaxation, and the interference effect on relaxation can be observed.

43. TROSY Effect is Field Dependent and Motion Dependent
Linewidth
Magnetic field strength
800 (kDa)
150 kDa
50 kDa
Optimal field strength: 1 GHz for amide NH;
600 MHz for CH in aromatic moieties ( MHz applicable).

44. NMR Structural study of larger proteins
Deuteration:
NMR Structural study of larger proteins
Deuteration is also an important techniques for NMR study of larger proteins (> 20kDa). It is achieved by raising the E. coli. in D2O medium (NT$ / 1L D2O).
Because of the significantly lower gyromagnetic ratio of 2H compared to 1H (g[2H] / g[1H] = 0.15), replacement of protons with deuterons removes contributions to proton linewidths from proton-proton dipolar relaxation and 1H-1H scalar couplings.
The effect of deuteration is similar with that of TROSY and both techniques are frequently used for NMR study of larger proteins.

45. The Sensitivity and Resolution Gain by TROSY and Deuteration
2H,15N-Gyrase-45 (45 kDa), 750 MHz
Wider and Wuthrich, Current Opinion in Structural Biology, 1999, 9:

46. Deuteration and 15N Specific-Labeling on Human Pancreatitis-Associated Protein (hPAP)
15N-Leu
15N-Tyr
15N-Ala

47. Investigate the Interactions between RNase and DNA
L62

48. Mapping the Binding Pocket
R37 Q1
V103 F6
H104
F105
V106
K96
T39
C94
I73 K5 K9
H10
I11
K33
V30
D18
I17
S87
N91
G107
K60
L62
R59
C97
K99
E98
0.09
0.05
0.2 ppm
Disappear
Mapping the Binding Pocket

49. Filtered NOESY can Get Inter-Molecule NOEs

50. Protein NMR Spectroscopy
NMR spectroscopy is a very powerful tool to study protein structure, protein modification, protein-ligand interaction, protein misfolding and protein folding. Although to understand the theory of NMR completely is a hard task, its application is easier to follow. Wish all of you find some thing interesting in your research life. If it’s NMR, just contact us.

53. 3D Structure Determination of Porcine β-Microseminoprotein (β-MSP)
Find NOE connectivities between N- and C-domains.

54. 3D Structure Determination of Porcine β-Microseminoprotein (β-MSP)
Structure refinement by NH residual dipolar couplings
1H Chemical Shift (ppm)
Isotropic solution
+ 5.3 mg/ml Pf1
15N Chemical Shift (ppm) C- N-
The RMSD of 20 NMR structures calculated with (in red) and without RDC restraints (in navy blue) superimposed in secondary structure regions is 0.73Å.

55. NMR Can Detect the Modification of Residues :
The Formation of Pyroglutamate
Gln
CBCA(CO)NH
Glu

56. NMR Can Detect the Modification of Residues :
The interactions between residues
We can further identify the residues that interact with Pyr1 from their chemical shift deviations.

57. Identify NOE Connectivity Between These Residues
Pyr1
V96 K9

58. NMR Solution Structure of hPAP

59. The Surface of hPAP
180
-0.3
0.3

60. Overview of Amyloid Fibril Formation of hPAP
Native Protein
Globular
Aggregates
(toxic)
Amyloid Fibril

61. Mapping the Core of hPAP Fibrils by H/D Exchange
15N-HSQC Spectra of hPAP in 90% DMSO
No D2O
With D2O

62. 1D Protein NMR spectrum H2O Ha Ha side-chain protons Methyl protons
Aromatic protons Ha
Backbone NH Ha
Indo NH of Trp